T7 rna polymerase mutants and their applications

Abstract

The present disclosure provides mutant T7 RNA polymerase proteins obtained through the introduction of one or more amino acid mutations (e.g., substitutions) at different sites based on the wild-type T7 RNA polymerase. Compared to the wild type T7 RNA polymerase, these mutants exhibited one or more of the following characteristics: lower levels of dsRNA by-products, higher product integrity, increased yield, or increased transcriptional activity. The mutant T7 RNA polymerase proteins of the present disclosure, with reduced levels of dsRNA by-products, are more suitable for in vitro transcription systems than compared to the wild-type T7 RNA polymerase. The significantly low levels of by-products and higher levels of purity allow for the requirements of RNA-based agents or mRNA vaccines to be readily met through simple purification, significantly reducing downstream purification complexity and costs. Additionally, the transcription products are of high quality and may have lower immune side effects.

1 . A mutant T7 RNA polymerase protein selected from the group of:
 a1: a protein comprising a substitution at one or at least two of the amino acid residues of a wild-type T7 RNA polymerase comprising the amino acid sequence set forth in SEQ ID NO:1, the amino acid residues being selected from the group of:   I6, I19, K60, N67, A70, N86, F162, V186, N233, A247, P277, A465, L651, or N764; or   a2: a protein comprising an amino acid sequence having at least 90% sequence identity to a mutant T7 RNA polymerase protein of a1, and whose activity and enzymatic properties are substantially the same as one or more of a mutant T7 RNA polymerase protein of a1.   
     
     
         2 . The mutant T7 RNA polymerase protein according to  claim 1 , wherein the protein further comprises a substitution at one or at least two of the amino acid residues selected from the group of F11, I82, D87, K180, V214, M369, Y457, H523, K610, A615, S686, or K740. 
     
     
         3 . The mutant T7 RNA polymerase protein according to  claim 1 , wherein the substituted protein further comprises one or more of an additional substitution, deletion, or addition of a mutant T7 RNA polymerase protein of  claim 1 , the protein having substantially the same activity and enzymatic properties as one or more of a mutant T7 RNA polymerase protein of  claim 1 . 
     
     
         4 . The mutant T7 RNA polymerase protein according to  claim 2 , wherein the protein comprises one or a combination of at least two of an amino acid substitution selected from the group of: I6G, F11L, I19T, K60I, N67S, A70Q, A70T, I82V, N86D, D87G, F162S, K180E, K180D, V186I, V214A, N233D, A247T, A247I, P277L, M369S, M369T, Y457H, A465T, H523R, K610R, A615T, L651M, S686G, K740R, or N764D. 
     
     
         5 . The mutant T7 RNA polymerase protein according to  claim 2 , wherein the protein comprises one or a combination of at least two of an amino acid substitution selected from the group of: I6G, F11L, I19T, K60I, N67S, A70Q, N86D, F162S, K180E, K180D, V186I, V214A, N233D, A247I, P277L, M369S, Y457H, A465T, H523R, L651M, or N764D. 
     
     
         6 . A mutant T7 RNA polymerase protein selected from the group of:
 b1: a protein comprising a substitution at one or at least two of the amino acid residues of a wild-type T7 RNA polymerase comprising the amino acid sequence set forth in SEQ ID NO:1, the amino acid residues being selected from the group of:   I6G, F11L, I19T, K60I, N67S, A70Q, A70T, N86D, F162S, K180E, K180D, V186I, V214A, N233D, A247T, A247I, P277L, M369S, Y457H, A465T, H523R, L651M, or N764D;   b2: a protein comprising at least two of the amino acid residues of a wild-type T7 RNA polymerase comprising the amino acid sequence set forth in SEQ ID NO:1, the amino acid residues being selected from the group of:   I82V, D87G, M369T, K610R, A615T, S686G, or K740R;   b3: the protein of b1 further comprising a substitution at one or at least two of the amino acid residues of a wild-type T7 RNA polymerase comprising the amino acid sequence set forth in SEQ ID NO:1, the amino acid residues being selected from the group of:   I82V, D87G, M369T, K610R, A615T, S686G, or K740R; or   b4: a protein comprising an amino acid sequence having at least 90% sequence identity to a mutant T7 RNA polymerase protein of b1, b2, or b3, and whose enzymatic activity and properties are substantially the same as one or more of a mutant T7 RNA polymerase protein of b1, b2, or b3.   
     
     
         7 . A mutant T7 RNA polymerase protein selected from the group of:
 c1: a protein comprising a substitution at one or at least two of the amino acid residues of a wild-type T7 RNA polymerase comprising the amino acid sequence set forth in SEQ ID NO:1, wherein the substitution is selected from the group of:
 (1) N86D/A615T; 
 (2) F162S; 
 (3) F162S/K180E; 
 (4) A247I; 
 (5) P277L; 
 (6) A70Q/F162S/K180E; 
 (7) N233D/A465T; 
 (8) D87G/N233D/V186I; 
 (9) A70T/P277L; 
 (10) N67S/I82V/Y457H/K610R/L651M/N764D; 
 (11) F162S/A247T; 
 (12) F162S/N233D/A247T; 
 (13) V214A; 
 (14) I19T/K180D/M369T; 
 (15) F162S/K180E/A247I; 
 (16) A70Q/V214A; 
 (17) A465T; 
 (18) F11L; 
 (19) K180E; 
 (20) M369S/S686G; 
 (21) K60I/A465T/K740R; 
 (22) H523R; 
 (23) A70T/K180D; 
 (24) I6G; 
 (25) A70Q/K180E; 
 (26) A70Q; 
 (27) V214A/A465T; or 
 (28) A70Q/A247T; or 
   c2: a protein comprising an amino acid sequence having at least 90% sequence identity to a mutant T7 RNA polymerase protein of c1, and whose activity and enzymatic properties are substantially the same as one or more of a mutant T7 RNA polymerase protein of c1.   
     
     
         8 . The mutant T7 RNA polymerase protein according to  claim 7 , wherein the substituted protein further comprises one or more of an additional substitution, deletion, or addition of a mutant T7 RNA polymerase protein of  claim 7 , the protein having substantially the same activity and enzymatic properties as one or more of a mutant T7 RNA polymerase protein of  claim 7 . 
     
     
         9 . The mutant T7 RNA polymerase protein according to  claim 1 , wherein the protein further comprises one or more of a tag or an enzyme cleavage site at one or both of the N-terminus or the C-terminus of the protein, and wherein the tag or the enzyme cleavage site does not substantially affect the activity and enzymatic properties of the mutant T7 RNA polymerase protein. 
     
     
         10 .- 11 . (canceled) 
     
     
         12 . The mutant T7 RNA polymerase protein according to  claim 1 , wherein the protein, as compared to a wild-type T7 RNA polymerase comprising the amino acid sequence set forth in SEQ ID NO:1, is characterized by producing during transcription one or more of a lower amount of dsRNA by-products, a higher product integrity, a higher yield, a higher transcriptional activity, or a lower cap analog usage. 
     
     
         13 . The mutant T7 RNA polymerase protein according to  claim 1 , wherein the protein is characterized by being capable of catalyzing co-transcriptional capping. 
     
     
         14 . A composition comprising the mutant T7 RNA polymerase protein according to  claim 1 , and one or more of an in vivo transcription reagent or in vitro transcription reagent. 
     
     
         15 . A transcription kit comprising a mutant T7 RNA polymerase protein according to  claim 1 . 
     
     
         16 . The transcription kit according to  claim 15 , wherein the transcription kit is a co-transcription capping kit. 
     
     
         17 . The transcription kit according to  claim 16 , further comprising a cap analog and a buffer system. 
     
     
         18 . (canceled) 
     
     
         19 . A method of generating a transcription product of a deoxyribonucleic acid (DNA) comprising contacting the DNA with a mutant T7 RNA polymerase protein according to  claim 1 . 
     
     
         20 . The method according to  claim 19 , further comprising contacting the transcription product with a capping enzyme to form a capped transcription product. 
     
     
         21 . The method according to  claim 19 , further comprising combining the transcription product or the capped transcription product with a pharmaceutically acceptable additive to produce a pharmaceutical dosage form. 
     
     
         22 . A method of generating a capped transcription product of a deoxyribonucleic acid (DNA) comprising:
 (i) mixing the DNA, a cap analog, and a mutant T7 RNA polymerase protein according to  claim 1 , in a buffer system; and   (ii) generating the capped transcription product through transcription of the DNA using the mixture of step (i).   
     
     
         23 . The method of  claim 22 , further comprising combining the capped transcription product with a pharmaceutically acceptable additive to produce a pharmaceutical dosage form.